Isolation of nucleic acid

ABSTRACT

The invention provides a method for isolating nucleic acid from a sample, said method comprising boiling said sample and allowing it to cool, and condensing the nucleic acid onto a high-surface area solid support, and in particular the use of such a method in the preparation of nucleic acid samples for subsequent amplification. The method has particular utility in the isolation of nucleic acid from aged, fixed or otherwise distressed samples.

The present invention relates to the isolation of nucleic acid,especially DNA, and in particular to a method for preparing nucleic acidsamples for subsequent use in amplification procedures.

Techniques for the amplification of nucleic acids have recentlyrevolutionised molecular biology and are now established as anindispensable tool in many procedures, for example in the detection anddiagnosis of genetic and infectious diseases, in forensic medicine, inthe identification of new genes or allelic variations or mutations, andin aiding routine genetic manipulations e.g. sequencing.

However, whilst new and improved amplification procedures arecontinually being developed, there are in certain cases particulardrawbacks which limit the utility of the technique. Thus for example,DNA-based diagnostic techniques relying on DNA amplification, e.g. bythe polymerise chain reaction (PCR), to detect the presence of microbialgenes have proved useful in detecting bacterial and viral infectiousagents and pathogenes and are rapidly acquiring importance.Nevertheless, in view of certain problems the techniques are notsuitable for all diagnostic uses. One major problem is thatamplification techniques such as PCR cannot be used directly on clinicalsamples, notably faeces or blood, which contain substances which inhibitthe amplification enzymes, e.g. polymerases. The presence of red bloodcells or haemoglobin presents a particular problem, and these generallyneed to be removed. A similar problem applies in the case of detectingmicrobial contamination in food samples, which also often containinhibitory substances.

For successful amplification, the sample either has to be diluted verymany times or the DNA has to be isolated and purified from the sample.In the former case, dilution to a degree sufficient to permitamplification frequently entails an unacceptable loss of sensitivity. Inthe latter case, nucleic acid purification techniques, involving forexample extraction with phenols, chloroform and alcohols are oftentedious, complicated and time consuming and may lead to loss of sampleDNA, which can be a problem if the sample is small.

PCR has also proved difficult to apply to samples which are old, whichhave been chemically treated (for example by fixing or embedding) orwhich are otherwise distressed. This significantly impairs the utilityof the technique in for example the analysis of fixed archival materialor of blood and tissue samples which are not fresh or which have notbeen stored under refrigeration. Fixation techniques now generally useddo not permit ready release of DNA suitable for the subsequentamplification using conventional techniques, and whilst improved, lessdamaging, fixation techniques are being developed, the situation is notentirely satisfactory. Complicated treatment procedures, e.g.deparaffinization of paraffin-embedded material, proteinase digestionetc are generally required and in many cases amplification cannot beachieved at all. This applies also in the case of aged ornon-refrigerated samples.

There therefore exists a need for an improved method for preparingnucleic acid for use in amplification procedures, which is quick andsimple to perform and which may be used on aged, non-refrigerated, fixedor otherwise treated or distressed samples. The present inventionaddresses this need.

We have now found that nucleic acid may be isolated from a sample in aform directly suitable for amplification by a simple and easy to performprocedure which involves boiling or heating the sample to a hightemperature and allowing it to cool, and depositing the nucleic acidonto a solid support. This procedure avoids many of the complicated andtime-consuming treatment steps of the prior art and, more importantly,can successfully be directly applied to clinical and otherblood-containing samples and to samples which are aged, non-refrigeratedor fixed, where previous techniques have proved unsuccessful. Moreparticularly, the invention is based on the surprising discovery thatwhen a sample is treated in this manner, nucleic acid released is ableto condense around the support, thereby allowing it to be separated fromthe sample, whilst at the same time, retaining the ability to act as atemplate in a subsequent amplification reaction.

According to one aspect, the present invention thus provides a methodfor isolating nucleic acid from a sample, said method comprising boilingsaid sample and allowing it to cool, and condensing the nucleic acidonto a high-surface area solid support.

As mentioned above, this method has particular utility in thepreparation of nucleic acid samples for amplification procedures sincethe resulting condensed nucleic acid samples can be used directly as thetemplate for amplification without requiring prior removal from thesupport.

The nucleic acid may be DNA, RNA or any modification thereof. Preferablyhowever the nucleic acid will be DNA, which may be genomic or cDNA, andsingle or double stranded. Where the method is used to prepare nucleicacid for amplification, it will preferably be double-stranded genomicDNA.

The sample may be any sample containing nucleic acid, but preferablywill be a clinical sample such as a blood, blood-derived, faeces ortissue sample or a sample which is aged or treated. Treated samplesinclude those which have been fixed, for example in formalin, acetone,alcohols, or any known or proprietary fixative e.g. Omnifix, orembedded, for example in paraffin wax or artificial or natural resins.Aged samples includes any sample which has not been freshly taken, orimmediately refrigerated. Such aged or treated samples thus include anysource of nucleic acid, plant or animal. In addition to archivalmaterial, which may be very many years old e.g. over one hundred years,or more preferably over 50 or 20 years old, such samples thereforeinclude samples of any biological tissue or fluid (e.g. blood, plasma,serum, organ or other tissue biopsies) which have not immediately beenrefrigerated or processed, e.g. clinical samples taken in remote areaswhich need to be transported and/or stored sometimes over a period ofdays, weeks or months, before they can be processed or analyzed.

This represents an important advantage of the present invention, as inthe past it has proved extremely difficult, if not impossible, toperform PCR or other amplifications on clinical samples, e.g. blood orbiopsies which have had to be transported, for example by post, to areference laboratory for analysis. Thus, advantageously, the presentinvention may be used when medical studies are conducted in remoteregions of the world, where specialised equipment and personnel arelacking, and where samples require fixation and/or storage for extendedperiods of time before analysis. Particularly advantageously, the needfor refrigeration may be avoided which is of significant benefit wheresamples need to be transported, e.g. by post,

Other samples on which the invention has been shown to work successfullyin preparing nucleic acid for amplification where previous methods havefailed, include hair roots and cells of the cheeklining, obtained bymouthwashes, scrapings, etc. The method of the invention has been shownto work particularly favourably on samples which contain fragmentednucleic acid. "Boiling" as used herein, includes heating of the sampleto high temperature, e.g. to at least 80° C., more preferably at least85 or 90° C.

The duration and temperature used in the boiling step is to some extentdependant on the nature and state of the nucleic acid containing sample.Chemically treated, e.g. formalin treated, or aged samples generallyrequire and can withstand more aggresive treatment than fresh cellsuspensions. In general, the boiling stage will conveniently be effectedfor 10 seconds to 1 hour, or more if convenient, for example 30 secondsto 30 minutes, or more particularly 3 to 15 minutes. Thus, for example,for most samples treatment at 94° C. for 10 minutes will normally allowsufficient nucleic acid isolation to permit effective amplification.

The support may be present with the sample during the heat treatment orit may be introduced subsequently, even days or hours after the samplehas been allowed to cool. For fresh cell suspensions it will generallybe convenient to introduce the support before, during or shortly afterthe boiling stage; however for samples where quantities of detritusseparate out as a result of the boiling stage it will generally bepreferred to introduce the support after such detritus has been removed.Thus for samples such as paraffin-embedded tissue, boiling produces aninhomogeneous mixture containing tissue and wax fragments. It ispreferred to separate off the nucleic acid containing liquid phase fromthese mixtures, e.g. by decanting or pipetting, after boiling andoptionally after cooling and then to introduce that liquid phase to thesupport to allow the nucleic acid condensation to occur.

The nucleic acid condensation (and preferably also the boiling andcooling steps) in the method of the invention is preferably carried outin a high-salt aqueous solution (e.g. having an osmolality equivalent tothat of at least 1 M NaCl aqueous solution).

Where condensation is effected by contacting the support with an alreadyboiled and cooled nucleic acid containing aqueous sample, such contactis preferably made for a period of some minutes, e.g. 1 to 60,especially 10 to 20 minutes to allow the condensation, i.e. the bindingof the nucleic acid to the support, to occur to an adequate extent.

The high-surface area support may be any known or conventional solidsupport presenting a high surface area for condensation of the nucleicacid. Such supports will generally have an irregular surface, and mayfor example be porous or particulate eg. particles, fibres, webs,sinters, or sieves. Condensation of the nucleic acid may take place onor around the support, or in it, if it has a porous structure forexample. Examples of suitable solid supports include microtitre wells,capillaries, fibres, filters and dipsticks, although particulatesupports are generally preferred, especially beads, as they, with theircaptured, "condensed" nucleic acid, may readily be used directly in asubsequent amplification step without any need to detach the condensednucleic acid from the support.

Conveniently, a particulate solid support used according to theinvention will comprise spherical beads. The size of the beads is notcritical, but they may for example be of the order of diameter of atleast 1 and preferably at least 2 μm, and have a maximum diameter ofpreferably not more than 10 and more preferably not more than 6 μm.Beads of diameter 2.8 μm and 4.8 μm have been shown to work well.

Monodisperse particles, that is those which are substantially uniform insize (e.g. size having a diameter standard deviation of less than 5%have the advantage that they provide very uniform reproducibility ofreaction. Monodisperse polymer particles produced by the techniquedescribed in U.S. Pat. No. 4,336,173 are especially suitable.

Non-magnetic polymer beads suitable for use in the method of theinvention are available from Dyno Particles AS (Lillestr.o slashed.m,Norway) as well as from Qiagen, Pharmacia and Serotec.

However, magnetic particles are particularly preferred as a solidsupport according to the invention, as they lend a number of advantages,Most notably, magnetic aggregation provides a quick, simple andefficient way of separating the particles following the condensationstep, and is a far less rigorous method than traditional techniques suchas centrifugation which generate shear forces which degrade nucleicacids.

Magnetic particles suitable for use as supports in the method of theinvention are available from Dynal, Advanced Magnetics Inc.,Biotechnologies Ltd., Amersham, Promega, Scigen, Advanced GeneticTechnologies and Seradyn.

Especially preferred are superparamagnetic particles, for example thosedescribed by Sintef in WO-A-83/03920, as magnetic aggregation andclumping of the particles during reaction can be avoided, thus ensuringuniform and nucleic acid abstraction.

The well-known magnetic particles sold by Dynal AS (Oslo, Norway) asDYNABEADS, are particularly suited to use in the present invention.Particular mention may be made of Dynabeads® M-450 and M-280.

It may be noted that the method of the invention works well despiteprevious studies using such magnetic particles having shown thatnon-specific binding of DNA and/or RNA to such surfaces is very low.

To improve the selectivity and performance of the technique, especiallywhere only minute quantities of a sample are available, oligonucleotideprobes specific for particular genes or nucleotide sequences mayadditionally be used, optionally together with stringent washing. Thus aspecific probe may be introduced into the sample, binding to a specifictarget sequence. To facilitate isolation of the target nucleic acid fromthe sample, the solid support may be provided with means for capture ofthe probe. Generally, this will be accomplished by providing each of theprobe and the support with one of a pair of corresponding affinitybinding partners, such that the probe and the support may be boundtogether selectively, and if desired, reversibly. Most conveniently theaffinity binding partner will comprise biotin and avidin/streptavidin,the biotin being bound to the probe and the avidin/streptavidin to thesupport. Both biotin-labelled oligonucleotide probes andstreptavidin-coated magnetic particles are commercially available (DynalAS).

Other binding partner systems may however be used, for exampleDNA-binding proteins binding to specific regions of the oligonucleotideprobe e.g. the lac repressor protein Lac I binding to a lac operator(LacOP) site which may be provided on the probe. Alternatively, theoligonucleotide probe may be labelled with a hapten such as digoxigenin,and the support may be provided with an anti-hapten antibody. Techniquesfor labelling oligonucleotides with haptens such as digoxigenin are wellknown in the art as are methods for attachment of antibodies to solidsupports.

Such a system is especially suited to the preparation of samplescontaining specific target nucleic acid sequences of interest, foramplification reactions. In particular we have shown that where priorart methods based upon isolation of target DNA for PCR by the use ofspecific biotin-labelled probes bound to streptavidin-coated magneticbeads have failed, e.g. in the case of blood containing samples, or agedor fixed samples, the method of the present invention has workedsuccessfully.

According to a further aspect, the present invention thus provides amethod of preparing a nucleic acid sample for in vitro amplification,said method comprising, sequentially or simultaneously, contacting saidsample with an oligonucleotide probe specific for a target nucleotidesequence within said sample, and boiling said sample, subsequentlyallowing the sample to cool, and condensing the nucleic acid onto a highsurface area solid support, wherein each of the oligonucleotide probeand solid support is provided with one of a pair of affinity bindingpartners, whereby the probe, and hence the target nucleotide sequence isbound to the support.

Alternatively viewed, this aspect of the invention provides a method ofamplification of nucleic acid within a sample, said method comprisingthe steps of (a) sequentially or simultaneously, contacting said samplewith an oligonucleotide probe specific for a target nucleotide sequencewithin said sample, and boiling said sample, subsequently allowing thesample to cool and condensing the nucleic acid onto a high surface areasolid support, wherein each of the oligonucleotide probe and solidsupport is provided with one of a pair of affinity binding partners,whereby the probe, and hence the target nucleotide sequence is bound tothe support, and (b) subjecting nucleic acid isolated by step (a) to anin vitro amplification reaction.

Conveniently, the sample may be allowed to cool in the presence of asolid support, whereby the nucleic acid condenses on or in the support.

Generally, as mentioned above the affinity binding partner pair willcomprise biotin/streptavidin. This binding partner system is commonlyused in molecular biology applications and many methods of incorporatingor attaching biotin and streptavidin to the probe/support respectively,are known. Thus for example biotin may be incorporated by andstreptavidin may be coated onto the support as discussed by Dynal AS intheir "Technical handbook, molecular biology". The length of theoligonucleotide probe is not critical but may conveniently lie in therange of 10-200 nucleotides, more preferably 15-50 nucleotides.

In the case where the solid support is in the form of particles, thefunctionalisation of the particles and subsequent attachment of probesis conveniently such that each particle carries 10³ -10⁶ probes or probebinding sites.

A number of in vitro amplification techniques have been developed andmay be used according to the present invention. PCR and itsmodifications, e.g. the use of nested primers, will however generally bethe principal technique to be used.

In classical PCR, two primers are required and these may either bespecific to a target DNA sequence of interest, or one or two standardPCR primers. This may necessitate introducing a hybridisation site for astandard PCR primer according to techniques well known in the art e.g.by restriction and ligation.

Nested PCR involves the use of two further so-called "inner" primers,which hybridise or "nest" between the first "outer" primer pair in asecond series of amplification cycles. The use of four separate primingevents results in increased specificity of the amplification reaction.

The nested primer technique has further been modified in the DIANA(Detection of Immobilised Amplified Nucleic Acids) system (see Wahlberget al., Mol. Cell Probes 4:285(1990)), in which the inner, second pairof primers carry, respectively, means for immobilisation to permitcapture of amplified DNA, and a label or means for attachment of a labelto permit recognition. This provides the dual advantages of a reducedbackground signal, and a rapid and easy means for detection of theamplified DNA.

Other amplification techniques worthy of mention include Self-sustainedSequence Replication (SSR), the Q-beta replicase amplification systemand the Ligase Amplification Reaction (LAR).

In SSR, primers are used which carry polymerase binding sites permittingthe action of reverse transcriptase to amplify target RNA or ssDNA.

In the Q-beta replicase system, an immobilized probe captures one strandof target DNA and is then caused to hybridise with an RNA probe whichcarries as a template region a tertiary structure known as MDV-1 for anRNA-directed RNA polymerase, normally Q-beta replicase.

LAR hybridises two oligonucleotide probes to adjacent positions on thetarget nucleic acid so that ligation, e.g. using T4 ligase, produces alonger sequence, which after strand separation, can function as atemplate for further hybridisations and ligations.

In the methods of the invention, the order in which the various stepsare performed is not critical and variations and modification arepossible. For example, the solid support may be added to the sampleprior to boiling, or after the boiling step. The primers/probes requiredfor amplification may likewise be added prior to the boiling step,immediately after boiling, or after the cooling condensation step.

The use of magnetic particles as solid supports particularly facilitatesthe washing and separation steps and has a significant advantage in thatall the reactions, including amplification, may be performed in onereaction vessel, thereby considerably simplifying the reaction processand avoiding loss of nucleic acid fragments adhering to the vesselwalls.

Boiling of the sample may take place in any known or conventional mediumknown in the art for manipulation of nucleic acids. Thus for examplemany typical buffers e.g. washing buffers, are known and can be used.Generally the use of high salt (e.g. 1-4M) aqueous media is preferred.Exemplary buffers include Tris-buffered saline solutions, and Dynal AS'sBinding and Washing buffer (10 mM Tris-HCl, 1 mM EDTA, 2.0M NaCl, pH7.5) is particularly suitable. Similar buffers may be used for anyintermediate washing steps which may be required. It has been foundfavourable to include salt, e.g. 1 to 4 M NaCl, preferably 2M NaCl, inthe boiling medium.

Incubation times for boiling may vary between 10 seconds and severalhours, more conveniently between 1 and 15 minutes. Thus for example,favourable results have been achieved by heating samples to atemperature of 80 to 100° C. for a period of 10 minutes, or in somecases, 3 to 5 minutes.

Cooling may take place simply by allowing the sample to stand at ambienttemperature, for example for a period of 3 to 20 minutes.

In the case where specific oligonucleotide probes are used, it may bepossible to control to some degree the degree of nucleic acidfragmentation and the level of nucleic acid denaturization andcondensation and hence the degree of specificity, by reducing theduration and/or temperature of the boiling step.

The methods of the invention has been found to work successfully withsample volumes of 0.1 μL to 100 mL. Generally small sample volumes of 1to 100 μL, e.g. 10 to 20 μL are preferred. In the case where particlesare used as solid support, amounts of 1 to 500 μg preferably 20 to 200μg may conveniently be used.

The ability of the method of the invention to be performed with suchsmall sample quantities is of particular benefit as the quantity ofsample available is often very limited, e.g. with needle biopsy andforensic samples or where it is important to keep the sample for furtherstudy.

Where an oligonucleotide probe is used to enhance specificity, amountsof 0.1 pmol to 50 pmol, e.g. 1 to 5 pmol may be used.

The components required to perform the method of the invention mayconveniently be supplied in the form of kits, and such kits form afurther aspect of the invention. In a preferred embodiment such a kitwould generally comprise the reagents needed to identify particular genesegments from any given sample type, and particular examples of suchkits would be for selective amplification of ras or HLA genes or theirmutations, e.g. K-ras and its 12/13 codon point mutations. Typicallysuch kits will include magnetic beads for nucleic acid condensation;aqueous medium, e.g. buffered saline, for the heat treatment step; 5'and 3' end primers, nucleic acid polymerase and restriction enzymes forPCR amplification and, if desired RFLP assay; and, optionally,oligonucleotide probes conjugated or conjugable to the beads (e.g.biotinylated probes which are conjugable to streptavidin coated beads)which hybridize to some or all of the gene sequence or to adjacentsequences.

As mentioned above, the method of the invention has a number of uses andapplications. These include for example the detection of pathogens,diseases and allelic variations. Clinical samples may be screened forepidemiological information. A major proposed use is in the analysis ofclinical samples taken at remote locations which are transported toreference laboratories; blood samples may for example be directly postedwithout refrigeration once nucleic acid condensation and plasma and cellfragment purging has taken place. Thus the samples may be treated onsite by the simple boiling step to condense the nucleic acid on to asolid support prior to flushing off sample other remnants and subsequenttransport.

The technique also has utility in forensic medicine, and in alldisciplines where sample or specimen storage is required e.g. inzoology, botany, conservation and evolutionary biology, the study ofbiological diversity or ecological processes.

The invention will now be described by the following non-limitingExamples, with reference to the drawings in which:

FIGS. 1a and 1b show

(a) the results of gel electrophoresis following restriction fragmentlength polymorphism (RFLP) analysis of the PCR products of Example 1:

Lane 1: Marker DNA.

Lane 2: PCR from magnetic isolated DNA from small amount of fixed tumourtissue.

Lane 3: PCR from 5 μl of liquid from boiled sample

Lane 4: PCR from 1 μg of mutated control DNA (positive control).

Lane 5: PCR without DNA (negative control);

(b) gel electrophoresis showing the results of solid phase sequencing ofthe mutated PCR product (ras gene mutation) (Lane 2 of FIG. 1(a))mutation indicated by arrow;

FIGS. 2a and 2b show

(a) the results of gel electrophoresis following restriction fragmentlength polymorphism (RFLP) analysis of the PCR products of Example 2:

Lane 1: Marker DNA.

Lane 2: PCR from magnetic isolated DNA from blood sample.

Lane 3: PCR from 5 μl of liquid from boiled sample.

Lane 4: PCR from 1 μg of control DNA (positive control).

Lane 5: PCR without DNA (negative control);

(b) gel electrophoresis showing the results of solid phase sequencing ofthe mutated PCR product (HLA-DQB gene sequence) (Lane 2 of FIG. 2(a)).

EXAMPLE 1

Identification of K-ras mutation from pancreatic cancer

Two 5 μm thick slides are cut from the paraffin block from aformalin-fixed paraffin-embedded needle-biopsy from a pancreatic cancerand added to 400 μL of Binding & Washing buffer (10 mM Tris-HCl, pH 7.5,1 mM EDTA, 2.0 M NaCl) containing 3 pmol of a biotinylatedoligonucleotide (e.g. 5'-B-ACTGA ATATA AACTT GTGGT AGTTG GACCT-3')complimentary to the 5'-end of the K-ras gene segment.

The tubes are incubated at 94° C. for 5 minutes and the contents mixedtwice on a vortex-mixer for 20 seconds during this incubation.

When the tubes have been cooled to ambient temperature in a thermalcycler for about 3 minutes, the liquid phase is pipettes off and mixedwith 20 μg of streptavidin coated paramagnetic beads (Dynabeads® M-280streptavidin, Dynal, Norway).

The mixture is left at ambient temperature for 15 minutes.

The beads are isolated by magnetic separation (Dynal MPC®-E, Dynal,Norway), and added as template to a K-ras specific PCR reaction-mix(e.g. using the 5' and 3' end primers 5'-ACTGA ATATA AACTT GTGGT AGTTGGACCT-3' and 5'-TCAAA GAATG GTCCT GGAAC-3', and Taq DNA polymerase) 5 μLof the boiled liquid, collected before the beads are added, is used astemplate in a parallel control procedure.

Three serial PCR's with two intermediate destructions of non-mutatedalleles by-a specific endonuclease (e.g. Bst NI) are performed.

For RFLP analysis the amplification products are digested, e.g. with BstNI and analysed using gel electrophoresis, Results are shown in FIG. 1a.A detectable amplification product (lane 2) is obtained from the PCRwhere beads carrying DNA served as template, but not when the bead freeboiled liquid is used (lane 3).

The PCR-product is identified as mutated by RFLP, and the nucleotidesequence was determined by solid-phase sequencing (FIG. 1b) indicatingthe presence of a point mutation in codon 12.

Conventional PCR amplification of and RFLP assays for ras oncogenes havebeen discussed in several publications and the primers and enzymesconventionally used may be used in the method of the invention. Suchpublications include Kahn et al. Amplifications 4:22-26 (1990), Chen etal. Anal. Biochem. 195:51-56 (1991), Kahn et al. Oncogene 6:1079-1083(1991), Haliassos et al. Nucleic Acids Research 17:3606 (1989) and Kumaret al. Oncogene 3:647-651 (1988).

EXAMPLE 2

HLA-typing of an old blood sample

10 μl of EDTA-blood, stored at ambient temperature for 7 days, was addedto 200 μl of Binding & Washing buffer (10 mM Tris-HCl, pH 7.5,m 1 mMEDTA, 2.0 M NaCl) mixed with 20 μg of paramagnetic beads (Dynabeads®M-450, Dynal, Norway). A parallel control sample was prepared withoutbeads.

The tubes were then incubated at 94° C. for 10 minutes and cooled toambient temperature.

The beads were isolated by magnetic separation (Dynal MPC®-E, Dynal,Norway), and added to a HLA-DQB specific PCR reaction-mix (e.g. usingthe 5' and 3' end primers DQ-AMP A: 5'-GCATG TGCTA CTTCA CCAAC G-Biotin3' and DQ-AMP B:5'-CAGGT AGTTG TGTCT GCACA C-3', and Taq DNApolymerase). 5 μl of the sample solution prepared without beads, wasused as template in a parallel control procedure.

A 30 cycle PCR was performed.

For RFLP analysis the amplification products were analysed using gelelectrophoresis. The results are shown in FIG. 2a.

A detectable amplification product (column 2) was obtained from the PCRwhere beads carrying DNA served as template, but not when the bead freeboiled liquid was used (column 3).

The nucleotide sequence was determined by solid-phase sequencing (FIG.2b).

General Method Steps

For use in nucleic acid fragment amplification, the method of theinvention may thus conveniently be effected using the following steps:

Step 1:

Nucleic acid release from sample by boiling, in the presence (Example 2)or absence (Example 1) of solid support and in the presence (Example 1)or absence (Example 2) of an oligonucleotide probe.

Step 2:

Nucleic acid binding to solid support.

a) Cooling in the presence of solid support (Example 2).

b) Adding solid support to the cooled liquid (Example 1) .

Step 3:

Isolation of solid support with bound nucleic acid (e.g. magneticseparation).

Step 4:

Solid support with bound nucleic acid used as template in a subsequentamplification reaction.

What is claimed is:
 1. A method for isolating nucleic acid from asample, said method comprising boiling said sample, cooling the boiledsample, allowing the nucleic acid in the liquid phase of the cooledsample to directly bind to a solid support comprising magneticparticles, and separating the solid support with the nucleic acid boundthereto from the remainder of said liquid phase.
 2. A method as claimedin claim 1 wherein the nucleic acid is DNA.
 3. A method as claimed inclaim 1 wherein the sample is a clinical sample.
 4. A method as claimedin claim 1, wherein the sample is fixed.
 5. A method as claimed in claim1, wherein the sample is aged.
 6. A method as claimed in claim 1,wherein the boiling step is effected by heating the sample to at least80° C.
 7. A method as claimed in claim 6, wherein the sample is heatedfor 10 seconds to 1 hour.
 8. A method as claimed in claim 1, wherein thesupport is added to the sample prior to or during the boiling step.
 9. Amethod as claimed in claim 1, wherein the support is added to the sampleafter the boiling step.
 10. A method as claimed in claim 1, wherein atleast one of the boiling, cooling, and contacting steps is carried outin a high salt aqueous solution.
 11. A method as claimed in claim 2,wherein the nucleic acid is genomic DNA.
 12. A method as claimed inclaim 3, wherein the nucleic acid is genomic DNA.
 13. A method asclaimed in claim 3, wherein the sample is a blood sample.
 14. A methodas claimed in claim 3, wherein the sample is a tissue sample.
 15. Amethod as claimed in claim 13, wherein the nucleic acid is genomic DNA.16. A method as claimed in claim 14, wherein the nucleic acid is genomicDNA.
 17. A method for isolating nucleic acid from a sample that is fixedor aged, said method comprising boiling the fixed or aged sample,cooling the boiled sample allowing the nucleic acid in the cooled sampleto directly bind to a solid support having a high surface areacomprising magnetic particles, and separating the solid support with thenucleic acid bound thereto from the remainder of the cooled sample. 18.A method as claimed in claim 17 wherein the nucleic acid is DNA.
 19. Amethod as claimed in claim 17, wherein the boiling step is effected byheating the sample to at least 80° C.
 20. A method as claimed in claim17, wherein the support is added to the sample prior to or during theboiling step.
 21. A method as claimed in claim 17, wherein the supportis added to the sample after the boiling step.
 22. A method as claimedin claim 17, wherein at least one of the boiling, cooling and contactingsteps is carried out in a high salt aqueous solution.
 23. A method asclaimed in claim 17, wherein the solid support is particulate.
 24. Amethod as claimed in claim 18, wherein the nucleic acid is genomic DNA.